The retinoblastoma tumor suppressor, RB, is inactivated in the majority of human cancers, resulting in growth advantage. While the underlying basis of inactivation encompasses a variety of mechanisms (e.g. deregulated phosphorylation, direct oncoprotein binding, point mutation) all of these results in disruption of RB assembled complexes. When active, RB is hypophosphorylated and assembles complexes that are capable of repressing transcription and inhibiting DNA replication. Mitogenic signaling cascades or oncogenic lesions stimulate phosphorylation of RB, thereby disrupting the assembled complexes and enabling progression through S-phase. In contrast, anti-mitogenic signals (e.g. DNA damage) prevent phosphorylation and maintain RB-repressor complexes, thus inhibiting cellular proliferation. Therefore, understanding RB-mediated replication control is germane both to regulated cell cycle progression (i.e. the interplay of mitogenic and anti-mitogenic signaling) and tumorigenesis. RB controls the expression and activity associated with several key factors requisite for DNA replication. Specifically, we have demonstrated that acute RB activation results in two events that result in the cessation of efficient DNA replication. First, RB inhibits expression of Cyclin A, a critical mediator of DNA replication. Attenuation of Cyclin A is causally associated with inhibition of PCNA activity that is required for DNA replication. In addition to this relatively rapid mechanism, long-term RB activation results in replicative exit and the loss of multiple components of the replication machinery (e.g. MCM2 and MCM7). Together, our prior studies put forth the hypothesis that RB-mediated replication control is a central means through which RB regulates proliferation and executes the appropriate response to DNA damage. This action of RB is achieved through two temporally distinct mechanisms. In Aim I, we dissect the rapid action of RB on replication control through the identification of possible direct mechanisms and through the mechanistic analyses of Cyclin A/PCNA signaling axis. In Aim II, we determine the influence of prolonged RB activation on replication competence and those replication complexes that demarcate functional origins. In both contexts (Aims I and II), we will determine how RB loss enables DNA replication following acute vs. prolonged DNA damage. Lastly, we will determine the action of RB in an in vivo model of physiologically regulated DNA replication and the consequence of RB loss in tumor progression. As such these studies evaluate the action of RB-mediated replication control as a means to limit proliferation, modify the response to DNA damage, and suppress tumorigenesis.